SOS1 gene from halophila that confers salt tolerance

Abstract

An SOS gene has been isolated through PCR cloning from Thellungiella halophila and is designated TSOS1. The polynucleotide encodes a transmembrane protein with similarities to plasma membrane Na⁺/H⁺ antiporters from bacteria and fungi. The invention encompasses the TSOS1 gene, the corresponding protein, closely related polypeptides that confer salt tolerance, and related polynucleotides that encode a polypeptide that has Na+/H+ antiporter activity.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

The subject application claims the benefit of priority to U.S. Ser. No. 60/628,096, filed on Nov. 15, 2004, which is incorporated herein in its entirety.

FILED OF THE INVENTION

The present invention relates to proteins and nucleic acids that provide to salt tolerance in plants, and in particular the TSOS1 gene from Thellungiella halophila.

BACKGROUND OF THE INVENTION

High concentrations of saline in soils inhibit plant growth and further inhibit agricultural productivity. Modem agricultural practices including irrigation are known to increase salt concentrations when the available irrigation water evaporates, leaving dissolved salts behind. In areas containing salty soils such as Southern California, Arizona, New Mexico and Texas, it has become particularly important to develop salt tolerant cultivars of agronomically important crops. Salty soils decrease the rate at which water will enter the roots due to the osmotic pressure of the solution. If the salt concentrations are sufficiently high, water will actually be withdrawn from the plant roots, eventually leading to plant death.

Thus, development of salt tolerant cultivars is required to offset effects of irrigation, and for utilization of marginal agricultural areas, and use saltier irrigation water.

Traditional plant breeding methods require long term selection and testing to identify new cultivars. Thus far, these methods have not yielded crop plants cultivars with substantially improved salt tolerance.

Excessive sodium ions (Na⁺) are toxic to plants because of adverse effects on cellular metabolism and ion homeostasis. Sodium ions in saline soils are toxic to plants due to their adverse effects on K+/Na+ homeostasis, cytosolic enzyme activities, photosynthesis and metabolism (Niu, et al. (1995) Plant Physiol. 109:735-742; Jacoby B. (1999) in Handbook of Plant and Crop Stress, ed. Pessarakli M (Marcel Dekker, NY), pp. 97-123)). Niu et al. (1995), supra, report three mechanisms that prevent accumulation of Na+ in the cytoplasm: restricted Na+ influx; active Na+ efflux and compartmentalization of Na+ in the vacuole. One example of restricted Na+ influx includes the wheat low affinity cation transporter LCT1 which may mediate Na+ influx into plant cells (Schachtman et al. (1997) PNAS 94:11079-84)). Another restriction on Na+ influx includes wheat high-affinity K+ transporter HKT1 which functions as a Na+-K+ co-transporter, which confers low-affinity Na+ uptake at toxic Na+ concentrations (Rubio et al. (1995) Science 270:1660-1663). Also, non-selective cation channels have been found to play important roles in mediating Na+ entry into plants (Amtmann et al. (1998) Adv. Bot. Res. 29:76-112)).

An example of Na+ compartmentalization in the vacuole includes the Arabidopsis thaliana ATNHX1 gene which encodes a tonoplast Na+/H+ antiporter (Gaxiola et al. (1999) PNAS 96:1480-85)).

Active Na+ efflux transporters have been found in fungi. In the yeast Saccharomyces cerevisiae, plasma membrane Na+-ATPases play a predominant role in Na+ efflux and salt tolerance (Haro et al. (1991) FEBS Lett. 291:189-191)). In the fungus Schizosaccharomyces pombe, Na+/H+ antiporters are more important for Na+ efflux and salt tolerance (Jia et al. (1992) EMBO J 11:1631-40)).

Arabidopsis is a glycophyte that is not very salt tolerant, but can adapt to elevated salt concentrations. Several Arabidopsis SOS mutants defective in salt tolerance have been characterized (Wu et al. (1996) Plant Cell 8:617-27; Liu et al. (1997) PNAS 94:14960-64; and Zhu et al. (1998) Plant Cell 10:1181-1191)). The SOS mutants are hypersensitive to high external Na+ or Li+ and also are unable to grow under very low external K+ concentrations (Zhu et al., (1998), supra)). The SOS mutants are defined as three loci, SOS1, SOS2 and SOS3 (Zhu et al. (1998), supra)). The SOS3 gene has homology to animal neuronal calcium sensors and the yeast calcineurin B subunit (Liu et al. (1998) Science 280:1943-45)). In yeast, mutations in calcineurin B lead to increased sensitivity of yeast cells to Na+ and Li+ concentration (Mendoza et al. (1994) J. Biol. Chem. 269:8792-96)). The SOS2 gene encodes a serine/threonine type protein kinase (Liu et al. (2000), PNAS, in press). Halfter et al. (2000) PNAS, in press, report that the SOS2 protein physically interacts with and is activated by SOS3, suggesting an SOS2/SOS3 regulatory pathway for Na+ and K+ homeostasis and salt tolerance in plants.

The SOS1 mutant is more sensitive to Na+ and Li+ stresses than the SOS2 and SOS3 mutant plants (Zhu et al. (1998), supra)). Double mutant analysis indicates that SOS1 functions in the same pathway as SOS2 and SOS3 (Liu et al. (1997), supra; and Zhu et al. (1998), supra)). The SOS1 protein may be a target for regulation by the SOS3/SOS2 pathway. The SOS1 gene has been cloned from Arabidopsis thaliana (U.S. Pat. No. 6,727,408). The SOS1 protein has Na+/H+ transporter activity and homology to Na+/H+ antiporters from bacteria and fungi. SOS1 transcript is up-regulated by NaCl stress. The SOS2 mutation abolishes SOS1 up-regulation in the shoot. In the SOS3 mutant, no SOS1 up-regulation is found in the shoot or root. SOS1 gene expression appears therefore to be regulated under NaCl stress by the SOS3/SOS2 regulatory pathway. (U.S. Pat. No. 6,727,408).

The salt cress, or Thellungiella halophila, can withstand dramatic salinity up to 500 mM NaCl and grow in salt far in excess of the capability of Arabidopsis. Salt cress has been suggested to be a favorable model for further study of salt tolerance because it has desirable life history traits (small size, short life cycle, self-pollination and high seed number), and favorable genetic traits (self-fertilization, a small genome, efficient transformation and mutagenesis). The salt cress genome is less than twice the size of Arabidopsis. EST analysis of hundreds of salt cress clones indicates 90-95% identities between Arabidopsis and salt cress cDNAs and amino acid sequences (Bressan et al. (2001) Plant Physiol. 127:1354).

A full-length cDNA microarray of Arabidopsis containing thousands of cDNAs including SOS1, has been used for expression profiling of salt cress genes. It was found that 6 genes were strongly induced in salt cress in response to high salinity stress, whereas 40 genes were identified as salt stress-inducible in Arabidopsis. The expression profiles of genes highly expressed in salt cress under normal growth conditions (not high salinity) were reported to resemble those Arabidopsis genes induced under abiotic stress (such as high salinity). It was suggested that salt cress constitutively overexpresses a large number of genes, including SOS1, even under unstressed conditions (Taji et al. (July 2004) Plant Physiol. 135:1697). Although Taji et al. report that an SOS1 gene is expressed in salt cress, they do not provide the sequence or report whether the salt cress SOS1 gene confers salt tolerance to salt cress.

Inan et al. (July 2004) Plant Physiol. 135:1718, report that the salt cress SOS1 gene is 84% homologous to the Arabidopsis SOS1 gene. This level of homology is less than the 90-95% homology observed between Arabidopsis and salt cress housekeeping genes.

SUMMARY OF THE INVENTION

An SOS gene has been isolated through PCR cloning from Thellungiella halophila and is designated TSOS1. The gene or polynucleotide encodes a transmembrane protein with similarities to plasma membrane Na⁺/H⁺ antiporters from bacteria and fungi. Existence of the predicted protein in Thellungiella halophila suggests that a plasma membrane-type Na⁺/H⁺ antiporter is generally essential for plant salt tolerance.

The isolated Thellungiella polynucleotide of SEQ ID NO: 1 encodes the contiguous amino acid sequence of SEQ ID NO: 3 that is a protein that effects Na⁺/H⁺ transporter activity.

In its various embodiments, the invention comprises:

(i) the polynucleotide of SEQ ID NO: 1;

(ii) a polynucleotide sequence having at least 85% homology to SEQ ID NO:1, wherein the homologous sequence encodes a polypeptide that has Na+/H+ transporter activity;

(iii) a polynucleotide sequence encoding the polypeptide of SEQ ID NO: 3, or encoding a homologous polypeptide having at least 84% homology to the polypeptide of SEQ ID NO: 3 and which has Na+/H+ transporter activity;

(iv) a polynucleotide having a sequence that has at least 85% homology to the sequence of (iii), and which encodes a polypeptide that has Na+/H+ transporter activity;

(v) a polynucleotide sequence that is a fragment of any of the sequences in (i)-(iv), wherein the fragment is at least 300 nucleotides in length, and wherein the fragment encodes a polypeptide that has Na+/H+ transporter activity; and

(vi) nucleic acid sequences complementary to or which hybridize under stringent conditions to any of the sequences of (i)-(v).

Regarding polynucleotide sequences described above under (ii), alternate embodiments include, with increasing preference, homologous polynucleotide sequences with minimum homology increasing incrementally by 1%, i.e., 86%, 87%, 88%, etc., up to and including 99%. Regarding the homologous polypeptide sequences described under (iii) above, alternate embodiments include, with increasing preference, homologous polypeptide sequences with minimum homology increasing incrementally by 1%, i.e., 85%, 86%, 87%, etc., up to and including 99%.

Regarding the polynucleotide fragment of (v) above, alternate embodiments include, with increasing preference, fragments with minimum lengths of 600, 900, 1200, 1500, 1800, 22100, 2400, 2700, 3000, 3300 nucleotides.

The invention also comprises a cassette comprising a heterologous promoter linked to any of the foregoing polynucleotides. In particular, the promoter can be an inducible promoter. The invention also comprises a vector which includes the cassette of heterologous promoter and foregoing polynucleotides.

Also included in the invention are host cells, including plant cells that comprise any of the foregoing polynucleotides, and transgenic plants comprising the foregoing polynucleotides. The transgenic plants can include crops, ornamentals, and trees, monocots and dicots. More specifically, this includes, without limitation, Arabidopsis thaliana, wheat, corn, peanut, cotton, oat, tomato, rice and soybean plants.

In another embodiment, the subject invention also comprises a method of making a transgenic plant with increased salt tolerance as compared to the plant's untransformed state. The transgenic plant is made by introducing any of the foregoing polynucleotides into the plant using methods known in the art.

The invention also includes the following polypeptides:

(i) the polypeptide of SEQ ID NO: 3;

(ii) a polypeptide with a sequence having at least 84% homology to SEQ ID NO: 3, and with increasing preference, homologous polypeptide sequences with minimum homology increasing incrementally by 1%, i.e., 85%, 86%, 87%, etc., up to and including 99%; wherein the homologous polypeptide has Na+/H+ transporter activity;

(iii) fragments of (i) or (ii), wherein said fragment is at least 100 amino acids in length, and with increasing preference, has a minimum length of 200, 300, 400, 500, 600, 700, 800, 900, 1000 and 1100 amino acids, and has Na+/H+ transporter activity.

The invention further comprises a method of identifying polynucleotides which encode a protein having Na+/H+ transporter activity, comprising: (a) identifying a polynucleotide in a nucleic acid sample or in a database that: (i) has at least 85% homology to SEQ ID NO: 1; (ii) has at least 85% homology to a polynucleotide that encodes SEQ ID NO.:3, or (iii) encodes a homologous polypeptide with at least 84% homology to SEQ ID NO: 3; (b) expressing the polynucleotide of (a) in a cell or transgenic plant; and (c) determining whether the cell or plant with the expressed polypeptide has Na+/H+ transporter activity. The polynucleotide that is identified can be substantially full-length relative to SEQ ID NO: 1, or a fragment that is at least 300 nucleotides in length. The identified fragment, with increasing preference, has a minimum length of 600, 900, 1200, 1500, 1800, 2100, 2400, 2700, 3000 or 3300 nucleotides.

The invention also comprises a method of identifying homologous or orthologous SOS1 polynucleotides in plants that are wild relatives of domesticated plant species. Such wild relatives are compatible with the domesticated crop species and are known to skilled artisans. The following table, table 1, taken from Colorado State University's Life Sciences Transgenic Crop program, sets forth compatible wild relatives. Wild relatives are generally considered to be more stress (cold, salt, etc.) tolerant. Those wild relatives in the table with an asterisk have been specifically reported to be more salt tolerant than their crop plants (see Wei et al. (2001) Plant Physiol. 125:1429 in relation to wheatgrass; and Latha et al. (2004) Annals of App. Biol. 144(2):177 in relation to breeding for salt tolerance).

TABLE 1

Crop species and compatible relatives.
Crop Species            Compatible relatives

alfalfa                 wild alfalfa
Medicago sativa         Medicago sativa
asparagus               wild asparagus
Asparagus officinalis   Asparagus officinalis
blueberry               wild blueberry
Vaccinium angustifolium Vaccinium angustofolium
burmuda grass           wild burmuda grass
Cynodon dactylon        Cynodon dactylon
carrot                  wild carrot
Daucus carota           Daucus carota
celery                  wild celery
Apium graveolens        Apium graveolens
chicory                 wild chicory
Chicorium intybus       Chicorium intybus
clover                  wild clover
Trifolium spp.          Trifolium spp.
corn                    wild relatives of corn
Zea mays subsp. mays    Zea mays subsp. mexicana
                        Zea mays subsp. parviglumis
                        Zea mays subsp. huehuetenangensis
                        Zea diploperennis
                        Zea perennis
                        Zea luxurians
cranberry               wild cranberry
Vaccinium macrocarpon   Vaccinium macrocarpon
foxtail millet          green foxtail
Setaria italica         Setaria viridis
lettuce                 wild lettuce
Lactuca sativa          Lactuca serriola
oats                    wild oats
Avena sativa            Avena fatua
oilseed rape, canola    wild radish
Brassica napus          Raphanus raphanistrum
                        wild brassicas
                        Brassica napus
                        Brassica campestris
                        Brassica juncea
quinoa                  wild quinoa
Chenopodium quinoa      Chenopodium berlandieri
radish                  wild radish
Raphanus sativus        Raphanus raphanistrum
rice                    red rice*
Oryza sativa            Oryza sativa
                        wild rice*
                        Porteresia coarctata
tobacco                 tobacco escaped from cultivation
Nicotiana tabacum       Nicotiana tabacum
sorghum                 Johnsongrass
Sorghum bicolor         Sorghum halapense
squash                  wild squash
Cucurbita pepo          Cucurbita texana
strawberry              wild strawberry
Fragaria X ananassa     Fragaria virginiana
sugar beets             wild beets
Beta vulgaris           Beta vulgaris
sunflower               wild sunflower
Helianthus annuus       Helianthus annuus
walnut                  California walnut
Juglans regia           Juglans hindsii
wheat                   jointed goatgrass*
Triticum aestivum       Aegilops cylindrical
                        wheat grass*
                        Lophopyrum elongatum

The wild relative SOS1 polynucleotide is identified by using probes or polynucleotide sequences from SEQ ID NO: 1 to screen polynucleotide libraries or databases of wild relative genomic or cDNA. The wild relative's SOS1 polynucleotide has at least 50% homology to SEQ ID NO: 1, and confers an improvement in salt tolerance when transformed into its counterpart domesticated plant. Also included are functional fragments of the wild relative's SOS1 polynucleotide, which are at least 300 nucleotides in length, and which confer improved salt tolerance on the counterpart transformed domesticated plant.

The subject invention also extends to a method of improving salt tolerance of domesticated plants by transforming with the wild relative's SOS1 polynucleotide or functional fragments thereof. These methods can be carried out with wild relatives of corn, rice, cotton, soybean, wheat and alfalfa.

Further, the subject invention encompasses the wild relative's SOS1 polynucleotide identified by the foregoing methods, and to domesticated plants that have been transformed with wild relative SOS1 to improve salt tolerance.

All references cited herein are incorporated by reference in their entirety.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art of molecular biology. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

Reference is made to standard textbooks of molecular biology that contain definitions and methods and means for carrying out basic techniques, encompassed by the present invention. See, for example, Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York (1982) and Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 3^rd Edtion (2001); Methods in Plant Molecular Biology, Maliga et al, Eds., Cold Spring Harbor Laboratory Press, New York (1995); Arabidopsis, Meyerowitz et al, Eds., Cold Spring Harbor Laboratory Press, New York (1994), Ausubel, et al., Current Protocols in Molecular Biology, Eds., Greene Publishing and Wiley-Interscience, New York (2000) and the various references cited therein.

The subject TSOS1 or salt cress SOS1 gene and its corresponding polypeptide provide a significant opportunity and advancement in agricultural genetic engineering due to the superior salt tolerance conferred by the SOS1 gene of Thellungiella halophyte as compared to the SOS1 gene of Arabidopsis. The discovery of the SOS1 gene provides methods for managing problems associated with increased salinity resulting from long-term irrigation, and methods for utilization of marginal agricultural areas and saltier irrigation water.

As discussed herein, the invention comprises the SOS1 gene of SEQ ID NO: 1, the polypeptide of SEQ ID NO: 3, and related polynucleotides and polypeptides that confer on a transformed plant cell or plant, a degree of salt tolerance that is improved relative to the salt tolerance exhibited by the untransformed plant cell or plant. Preferably, the improvement in salt tolerance relative to the untransformed plant or cell is at least 10%, 25% or more. Alternatively, the salt tolerance conferred by the homologous polynucleotide or polypeptide to the transformed plant or plant cell is substantially the same as that which is obtained with TSOS1 of SEQ ID NO: 1 and/or the polypeptide of SEQ. ID NO: 3.

Related molecules containing the subject polynucleotides are also included within the scope of the invention. Thus, cassettes comprising the polynucleotides and a heterologous promoter are included. Also, vectors comprising the polynucleotides and/or cassettes are within the scope of the invention. Further, host cells or plant cells, and transgenic plants are considered to be within the scope of the invention. Cassettes and vectors can be synthesized using methods known in the art, and can be transformed into plant cells and plants using methods known in the art.

A polynucleotide that falls within the scope of the present claims can be identified by: (a) identifying a polynucleotide in a nucleic acid sample or in a database that: (i) has at least 85% homology to SEQ ID NO: 1; (ii) has at least 85% homology to a polynucleotide that encodes SEQ ID NO.:3, or (iii) that encodes a homologous polypeptide having at least 84% homology to the polypeptide of SEQ ID NO: 3; (b) expressing the polynucleotide of (a) in a cell or transgenic plant; and (c) determining whether the expressed polypeptide has Na+/H+ transporter activity.

The subject method also comprises a method for identifying homologous or orthologous SOS1 polynucleotides in plants that are wild relatives of domesticated plant species. In this context, a “domesticated plant” is any plant that has been cultivated by humans as a crop. For example, domesticated plants include corn, rice, soybean, cotton, alfalfa, wheat and the like, grown for human or animal consumption. It also includes flowering or other decorative plants grown for aesthetic or landscaping purposes. “Wild relatives” of domesticated plants are those plants that are wild ancestors of, or otherwise related, to the domesticated plants. Wild relatives of domesticated plants are known to persons of skill in the art. For example, teosinte is a wild relative and ancestor of domesticated corn. Wild relatives useful in the subject invention are those which exhibit greater salt tolerance than that exhibited by the domesticated plant.

To identify a homologous SOS1 polynucleotide in a wild, salt tolerant relative, a polynucleotide probe of at least 20 nucleotides of SEQ ID NO: 1 is used to screen mRNA transcripts, cDNA or genomic DNA from the wild, salt tolerant relative of the domesticated plant. In the alternative, the sequence of SEQ ID NO: 1, or a polynucleotide sequence of at least 20 nucleotides thereof, is used to “blast” a database containing genomic, cDNA or mRNA transcript sequences of the wild, salt tolerant relative, to identify the homologous SOS1 polynucleotide in the wild, salt tolerant relative. In a preferred embodiment, the homologous SOS1 polynucleotide has at least 50% homology to SEQ ID NO: 1. It is increasingly preferred that the minimum homology of the wild relative's SOS1 polynucleotide be 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%, when compared to SEQ ID NO: 1.

Next, the wild relative's homologous SOS1 polynucleotide or a functional fragment thereof is transformed into the domesticated plant using methods known in the art. The transformed domesticated plant is examined to determine whether it exhibits salt tolerance that is improved relative to the salt tolerance of the untransformed domesticated plant. A wild relative's homologous SOS1 polynucleotide that improves salt tolerance of a domesticated plant is considered to fall within the scope of the invention.

A “functional fragment” of the wild relative's SOS1 polynucleotide has a minimum length of at least 300 nucleotides, and with increasing preference, a minimum length selected from the group consisting of 600, 900, 1200, 1500, 1800, 2100, 2400, 2700, 3000, and 3300 nucleotides. The functional fragment retains the ability to confer salt tolerance on the transformed domesticated plant that is improved relative to the untransformed domesticated plant

The subject invention also comprises a method for improving the salt tolerance of the domesticated plant. This is accomplished by identifying the wild relative's homologous SOS1 polynucleotide or functional fragment thereof, as discussed above, and transforming the wild relative's SOS1 polynucleotide or functional fragment into the domesticated plant, to produce a transformed domesticated plant with improved salt tolerance.

The foregoing methods of identifying SOS1 polynucleotides in wild, salt tolerant relatives, and using the SOS1 polynucleotides to enhance salt tolerance of domesticated species, can be carried out with wild relatives of corn, rice, cotton, soybean, wheat and alfalfa.

Example 2 exemplifies the foregoing methods. Salt cress is considered a wild relative of Arabidopsis Columbia. The Arabidopsis SOS1 gene was used to identify homologous salt cress SOS1 gene of SEQ ID NO: 1. The polynucleotide of SEQ ID NO: 1 was then used to transform the Columbia plants. As is demonstrated in Example 2, the transformed Columbia plants exhibit improved salt tolerance relative to the untransformed control Columbia plants. In fact, the Columbia plants transformed with salt cress SEQ ID NO: 1 polynucleotide was shown to have substantially greater salt tolerance as compared to the Columbia plants transformed with the Arabidopsis SOS1 gene, regardless of concentration of the NaCl solution.

The subject invention also extends to the wild relative's SOS1 polynucleotide which has been found by the foregoing methods to improve salt tolerance of the domesticated plant. The wild relative's domesticated relation can be corn, rice, cotton, soybean, wheat, alfalfa or the like.

The subject invention also extends to domesticated plants with improved salt tolerance obtained by transforming the plant with its wild relative's SOS1 polynucleotide. Again, such transformed domesticated plants can include corn, rice, cotton, soybean, wheat, alfalfa and the like.

Provided below are definitions of terms used herein:

“Enhancement” of a protein means increasing the intracellular activity of one or more proteins/enzymes in a plant cell and/or plant which are encoded by the corresponding DNA. Enhancement can be achieved with the aid of various manipulations of the cell. In order to achieve enhancement, particularly over-expression, the number of copies of the corresponding gene can be increased, a strong promoter can be used, or the promoter- and regulation region or the ribosome binding site which is situated upstream of the structural gene can be mutated. Expression cassettes which are incorporated upstream of the structural gene act in the same manner. In addition, it is possible to increase expression by employing inducible promoters. A gene can also be used which encodes a corresponding protein/enzyme with a high activity. Expression can also be improved by measures for extending the life of the mRNA. Furthermore, protein/enzyme activity as a whole is increased by preventing the degradation of the protein/enzyme. Moreover, these measures can optionally be combined in any desired manner. These and other methods for altering gene activity in a plant are known as described, for example, in Methods in Plant Molecular Biology, Maliga et al, Eds., Cold Spring Harbor Laboratory Press, New York (1995).

A gene can also be used which encodes a corresponding or variant protein/enzyme with a high activity. Preferably the corresponding protein/enzyme has a greater activity than the native form of the protein/enzyme, more preferably at least in the range of 5, 10, 25% or 50% more activity, most preferably more than twice the activity of the native or wild type enzyme.

“Fragment” refers to a portion of the polynucleotide sequence of SEQ ID NO:1 or homologs or degenerate variants thereof, or a portion of the polypeptide sequence of SEQ ID NO: 3. In the case of a polynucleotide sequence, the fragment is at least 300 nucleotides in length and encodes a polypeptide that has Na+/H+ transporter activity. The polynucleotide fragment, with increasing preference, has a minimum length of 600, 900, 1200, 1500, 1800, 2100, 2400, 2700, 3000 or 3300. In the case of a polypeptide sequence, the fragment is at least 100 amino acids in length and has Na+/H+ transporter activity. The polypeptide fragment, with increasing preference, has a minimum length of 200, 300, 400, 500, 600, 700, 800, 900, 1000 or 1100.

The SOS1 protein contains C-terminal and transmembrane N-terminal regions. The C-terminal region (about 100 amino acids) is cytosolic and undergoes phosphorylation reactions, while the N-terminal transmembrane region mediates Na+/H+ transport. This structure is similar to that reported for Arabidopsis SOS1 protein (Shi et al. (2000) PNAS 97:6896). Correlation of the subject SOS1 protein to the Arabidopsis SOS1 protein and other Na+/H+ antiporters reveals amino acids critical for function, and which should not be substituted (or only conservatively substituted) in functional homologs of the subject invention. Functional fragments of the subject invention can be selected according to information available from such comparative studies.

A “homologous” polynucleotide refers to a polynucleotide sequence that has, as compared to the polynucleotide sequences of the invention, at least 85% homology, and with increasing preference, a minimum homology percentage increasing incrementally by 1%, i.e., 86%, 87%, etc., up to and including 99%. According to the invention, a “homologous” protein is to be understood to comprise polypeptides which contain an amino acid sequence having at least 84% homology to the SOS1 polypeptide of SEQ ID NO: 3, and with increasing preference, a minimum homology percentage increasing incrementally by 1%, i.e., 85%, 86%, etc. up to and including 99%.

Homology, sequence similarity or sequence identity of nucleotide or amino acid sequences may be determined conventionally by using known software or computer programs such as the BestFit or Gap pairwise comparison programs (GCG Wisconsin Package, Genetics Computer Group, 575 Science Drive, Madison, Wis. 53711). BestFit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482-489 (1981), to find the best segment of identity or similarity between two sequences. Gap performs global alignments: all of one sequence with all of another similar sequence using the method of Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970). When using a sequence alignment program such as BestFit, to determine the degree of sequence homology, similarity or identity, the default setting may be used, or an appropriate scoring matrix may be selected to optimize identity, similarity or homology scores. Similarly, when using a program such as BestFit to determine sequence identity, similarity or homology between two different amino acid sequences, the default settings may be used, or an appropriate scoring matrix, such as blosum45 or blosum80, may be selected to optimize identity, similarity or homology scores.

“Increased salt tolerance” means that the plant cell or plant exhibits increased resistance to or tolerance of elevated salinity in the water and/or soil. This is manifested by phenotypic characteristics known to the person skilled in the art including longer life span and apparent normal growth and function of the plant even during elevated salinity. Salt tolerance is measured by methods known in the art such as those described in Inan et al. (July 2004) Plant Physiol. 135:1718, including without limitation, NaCl shock exposure or gradual increase NaCl concentration. An example of NaCl shock exposure is exposure of 3-week-old seedlings to irrigation water of 500 mM NaCl every other day. An example of gradual increase in NaCl exposure is exposure of 3 week-old seedlings to irrigation water with 75 mM NaCl for 5 days, followed by increases to 150, 200, 300, 400 and 500 mM on days 5, 9, 16, 22 and 26 (Inan et al. (2004), supra). Root growth and shoot growth or other parameters such as seed production can be measured as an indication of salt tolerance.

“Introduction of the polynucleotide into the plant” refers to any of a number of methods known to the skilled artisan for introduction of heterologous polynucleotide into a plant cell or plant. Such methods are described without limitation in the Examples.

“Isolated” means separated from its natural environment.

“Plant” refers to whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds and plant cells and progeny of same. The class of plants which can be used in the methods of the invention is generally as broad as the class of higher plants amenable to transformation techniques, including both monocotyledonous and dicotyledonous plants. Preferred plants include rice, corn, wheat, cotton, peanut, oat, tomato, alfalfa, canola, sunflower and soybean, and all other ornamental and forestry plants.

“Polynucleotide” refers in general to polyribonucleotides and polydeoxyribonucleotides, and can denote and unmodified RNA or DNA or a modified RNA or DNA.

“Polypeptides” means peptides or proteins which contain two or more amino acids which are bound via peptide bonds.

“Polypeptides that have Na+/H+ transporter activity” refers to polypeptides or proteins that confer salt tolerance on a transformed plant cell or plant that is improved relative to the untransformed plant cell or plant.

“Polypeptides that substantially retain TSOS1 Na+/H+ transporter activity” refers to polypeptides or proteins that have at least 84% homology to SEQ ID NO: 3, and which confer salt tolerance on a plant cell or plant that is substantially the same as that provided by the TSOS1 polypeptide of SEQ ID NO. 3.

As mentioned above, salt tolerance can be measured by methods known in the art such as those described in Inan et al. (July 2004) Plant Physiol. 135:1718, including without limitation, growth of roots or shoots during and following NaCl shock exposure or a gradual increase in NaCl concentration. Salt tolerance conferred by a homologous polynucleotide or polypeptide is “substantially the same” as that provided by the SOS1 polypeptide of SEQ ID NO: 3, if it provide at least 80% or 90% of the salt tolerance provided by the TSOS1 of SEQ ID NO: 3 in the same plant cell or plant under the same experimental conditions. The salt tolerance that is conferred by the homologous polypeptide in question can be the result of expression or overexpression of a single or multiple corresponding polynucleotides.

“Stringent conditions” or “stringent hybridization conditions” includes reference to conditions under which a polynucleotide will hybridize to its target sequence, to a detectably greater degree than other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified which are 100% complementary to the probe (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing).

Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° Celsius for short probes (e.g., 10 to 50 nucleotides) and at least about 60° Celsius for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37 degrees Celsius, and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55 degrees Celsius. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37 degrees Celsius, and a wash in 0.5× to 1×SSC at 55 to 60 degrees Celsius. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37 degrees Celsius, and a wash in 0.1×SSC at 60 to 65 degrees Celsius.

Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA—DNA hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl, Anal. Biochem., 138:267-284 (1984): Tm=81.5oC.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. Tm is reduced by about 1 degree Celsius for each 1% of mismatching; thus, Tm, hybridization and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with approximately 90% identity are sought, the Tm can be decreased 10 degrees Celsius. Generally, stringent conditions are selected to be about 5 degrees Celsius lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4 degrees Celsius lower than the thermal melting point (Tm); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10 degree Celsius lower than the thermal melting point (Tm); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20 degrees Celsius lower than the thermal melting point (Tm). Using the equation, hybridization and wash compositions, and desired Tm, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a Tm of less than 45 degrees Celsius (aqueous solution) or 32 degrees Celsius (formamide solution) it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Current Protocols in Molecular Biology, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (2000).

EXAMPLES

Example 1

Plant Transformation Methods

A DNA segment identified in this disclosure is isolated, and combined with at least a promoter functional in a plant cell to provide a recombinant expression cassette. An expression cassette comprising a recombinant DNA segment is subcloned into any standard expression vector by methods known to those of skill in the art. Suitable expression vectors include plasmids that autonomously replicate in prokaryotic and/or eukaryotic cells. The expression vector is introduced into prokaryotic or eukaryotic cells by currently available methods such as microprojectile bombardment, tungsten whiskers, and liposomes. The vector is introduced into prokaryotic cells such as E. coli or Agrobacterium. Transformed cells are selected typically by using a selectable screener marker encoded on the expression vector. A method to generate transgenic monocot plants is described in U.S. Pat. No. 6,281,411, the disclosure of which is herein incorporated by reference.

The expression vector or cassette is introduced into plant cells that include both monocot and dicot plants. Plant cells, or tissue, or organs useful for transformation include flowers, callus, immature embryos, meristematic tissue, gametic tissue, or cultured suspension cells. Other recombinant DNA molecules encoding proteins that enhance plant transformation may also be introduced. The transformed plant cell is regenerated into a plant and the plant is tested for its ability to grow or thrive under stress conditions, such a high salinity, reduced water availability, and adverse temperatures. Depending on the type of plant, the level of gene expression, and the activity of the protein encoded by the recombinant DNA segment, introduction of the recombinant DNA into the plant confers the phenotype of tolerance or resistance to the stress conditions to the plant.

The introduced recombinant DNA segment is expressed in the transformed plant cells and stably transmitted, somatically and sexually, to the subsequent cells produced. The vector is capable of introducing, maintaining, and expressing a recombinant DNA segment in plant cells, wherein the recombinant DNA is obtained from a variety of sources, including to plants, animals, bacteria, fungi, yeast, or virus. Additionally, the vector is introduced into a wide variety of plant cells.

Introduction and expression of foreign genes in dicotyledonous plants such as Arabidopsis, tobacco, potato, and alfalfa has been accomplished by using the T-DNA of the tumor inducing (Ti) plasmid of Agrobacterium tumefaciens. Through recombinant DNA techniques, a wide variety of foreign DNAs can be inserted into the T-DNA in Agrobacterium. Following infection by the bacterium containing the recombinant Ti plasmid, the foreign DNA is inserted into the host plant chromosomes, thus producing a genetically engineered cell and then a genetically engineered plant. Another approach is to introduce root-inducing (Ri) plasmids as gene vectors. Additionally, using the Ti plasmid as a model system, it may be possible to artificially construct transformation vectors for monocot plants. Ti plasmids may also be introduced into monocots by artificial methods such as microinjection, or fusion between monocot protoplasts and bacterial spheroplasts containing the T-region, which can then be integrated into the plant nuclear DNA. The selectable marker and heterologous gene expression cassettes described herein may be placed in a suitable expression vector designed for operation in plants. Suitable vectors are describe in U.S. Pat. Nos. 5,888,789 and 5,889,189, the disclosures of which are herein incorporated by reference.

Transformation of plant cells with recombinant DNA segments may also be accomplished by introducing a recombinant DNA into other nucleic acid molecules that can transfer the inserted DNA into a plant genome (i.e., plant pathogens such as DNA viruses like CaMV or gemini viruses, RNA viruses, and viroids); DNA molecules derived from unstable plant genome components like extrachromosomal DNA elements in organelles (i.e., chloroplasts or mitochondria) or nuclear-encoded controlling elements; DNA molecules from stable plant genome components (i.e., origins of replication and other DNA sequences which allow introduced DNA to integrate into the organellar or nuclear genomes and to replicate normally, to autonomously replicate, to segregate normally during cell division and sexual reproduction of the plant and to be inherited in succeeding generations of plants) or transposons.

A recombinant DNA may be delivered into plant cells or tissues directly by microorganisms with infectious plasmids, infectious viruses, the use of lipsomes, microinjection by mechanical or laser beam methods, by whole chromosomes or chromosome fragments, electroporation, and microprojectile bombardment. For example, callus cells or any other suitable tissue derived from the members of a desired plant species are transformed with an expression cassette that includes at least the recombinant DNA segment of interest, a selectable marker, and a suitable promoter using a variety of standard techniques (i.e., electroporation, Agrobacterium, protoplast fusion, or microparticle bombardment). A suitable transcription regulatory region (promoter) includes a plant promoter whose transcription is regulated in a response to various stress responses such as drought, salinity, and high temperature.

Suitable promoters include plant stress inducible promoter, viral coat protein promoter, a tissue-specific promoter, a monocot promoter, a ubiquitin promoter, a CaMV 35S promoter, a CaMV 19S promoter, a nos promoter, and Adh promoter, a sucrose synthase promoter, a tubulin promoter, a napin promoter, an actin promoter, a cab promoter, a PEPCase promoter, a 7S-alpha-conglycinin promoter, and R gene complex promoter, a tomato E8 promoter, a patatin promoter, a mannopine synthase promoter, a soybean seed protein glycinin promoter, a soybean vegetative storage protein promoter, a bacteriophage SP6 promoter, a bacteriophage T3 promoter, a bacteriophage T7 promoter, a PM promoter, a root-cell promoter, an ABA-inducible promoter, a turgor-inducible promoter, and any other stress-inducible promoters. Stress inducible promoters are particularly helpful. Exemplary transiently activated stress-inducible plant promoters are described in U.S. Pat. No. 6,414,221, the disclosure of which is herein incorporated by reference.

Example 2

Transformation of Arabidopsis with Salt Cress and Arabidopsis SOS1 Genes

Arabidopsis plants (Columbia) were grown in the greenhouse for 3 weeks in normal nutrient solution (Zhu et al. (2005) PNAS 102:9966) and then watered for ten days with the nutrient solution containing NaCl in concentrations indicated below. After 10 days, plants were re-watered with nutrient solution without any NaCl and the percent surviving plants were counted after two weeks. One hundred and fifty plants of each treatment and genotype were used in the experiment. Overexpression of SOS1-A and SOS1-SC salt cress was determined by RT-PCR and Northern blot analysis before and after salt treatment. Both SOS1-A and SOS1-SC plants accumulated 4 to 20 times more SOS1 transcript than control Columbia plants in three replicated experiments.

TABLE 2
Percent survival at various sodium chloride concentrations.
	NaCl
	0 mM	100 mM	200 mM	300 mM
	% Survival
Control	100	50	3	0
SOS1 (SOS1-A)	100	65	40	10
Arabidopsis
SOS1 (SOS1-SC)	100	95	80	50
Salt Cress

The foregoing results indicate that the salt cress SOS1 gene is superior to the SOS1 gene of Arabidopsis in conferring salt tolerance to transformed Arabidopsis Columbia plants.

These results are consistent with the report of Vera-Estrella et al. (2005) Plant Physiol. Preview, published after the priority date of the subject application. Vera-Estrella et al. describe how expression of SOS1 protein increases in salt cress leaves and roots after treatment of the plants with either 200 or 400 mM NaCl, indicating that the stress response even in salt cress is mediated (at least in part) by the SOS1 gene and polynucleotide.

The foregoing examples are intended to be illustrative and not exclusive.

Claims

1. An isolated polynucleotide having a sequence selected from the group consisting of: a) the nucleic acid sequence of SEQ ID NO: 1; b) a nucleic acid sequence having at least 95% sequence identity to the SEQ ID NO: 1, and encoding a polypeptide that has Na+/H+ transporter activity; and c) a nucleic acid sequence encoding the polypeptide of SEQ ID NO: 3, or encoding a polypeptide having at least 95% sequence identity to the polypeptide of SEQ ID NO: 3, and which has Na+/H+ transporter activity.

2. A cassette comprising a heterologous promoter operably linked to the polynucleotide of claim 1.

3. The cassette of claim 2, wherein the promoter is an inducible promoter.

4. A vector comprising the cassette of claim 2.

5. A host cell comprising the cassette of claim 2 and wherein the host cell expresses the polynucleotide.

6. A plant cell comprising the cassette of claim 2 and wherein the plant cell expresses the polynucleotide.

7. A plant organ or seed comprising the cassette of claim 2 and wherein said plant organ or seed expresses the polynucleotide.

8. A transgenic plant comprising the cassette of claim 2 and wherein the transgenic plant expresses the polynucleotide.

9. A progeny of the transgenic plant of claim 8, wherein the progeny comprises said polynucleotide.

10. The transgenic plant of claim 8 selected from the group consisting of Arabidopsis thaliana, wheat, corn, peanut, cotton, oat, tomato, rice, alfalfa, canola, sunflower and soybean plants.

11. A method of making a transgenic plant with increased salt tolerance as compared to the plant's untransformed state, comprising introducing the polynucleotide of claim 1 into said plant and expressing said polynucleotide that confers salt tolerance.

12. An isolated polynucleotide having a nucleic acid sequence which is fully complementary to the isolated polynucleotide of claim 1.